Method of treating cancerous tumors with a dendritic-platinate drug delivery system

ABSTRACT

Dendritic polymer conjugates which are useful drug delivery systems for carrying platinum containing anti-tumor agents to malignant tumors are prepared by obtaining a dendritic polymer having functional groups which are accessible to a platinum containing compound capable of interacting with the functional groups, contacting the dendritic polymer with the platinum containing compound, and reacting the dendritic polymer with the platinum containing compound. The dendritic polymer platinates may be administered intravenously, orally, parentally, subcutaneously or topically to an animal having a malignant tumor in an amount which is effective to inhibit growth of the malignant tumor. The dendritic polymer platinates exhibit high drug efficiency, high drug carrying capacity, good water solubility, good stability on storage, reduced toxicity, and improved anti-tumor activity in vivo.

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional application of application Ser. No. 09/111,232,filed Jul. 7, 1998, which is a continuing application of provisionalapplication Serial No. 60/051,800, filed Jul. 7, 1997, which is nowabandoned.

FIELD OF THE INVENTION

This invention relates to the treatment of cancer in animals, especiallyhumans, using dendritic polymer conjugates.

BACKGROUND OF THE INVENTION

The prospect of using dendritic polymers as carriers for drug deliveryhas been previously proposed on account of the unique structure andcharacteristics of these polymer molecules. More specifically, it hasbeen proposed that the external surface functionality and interiormorphological characteristics of dendritic polymer molecules appear tobe very promising for developing new methods for controlling drugrelease and targeted drug delivery systems. However, relatively littlework has been done in specific areas of drug delivery. In particular,the use of dendritic polymers as effective carriers for specificanti-tumor agents has not heretofore been demonstrated.

Certain platinum containing compounds, particularly carboplatin andcisplatin, have been used in the treatment of ovarian cancer, lungcancer, testicular cancer, breast cancer, stomach cancer and lymphoma.However, because of the non-specific toxicity and poor water solubilityof these platinum containing compounds, the use of carboplatin andcisplatin has been relatively limited.

In order to overcome the non-specific toxicity and water solubilityproblems associated with cisplatin and carboplatin, it has been proposedto use linear polymers as carriers for these drugs. However, the use oflinear polymers as carriers in drug delivery systems has severaldisadvantages. A major disadvantage with linear polymer drug carriers isthat they are heterogenous, polydisperse compositions containing variousdifferent molecular weight polymer molecules. Because linear polymercompositions are not comprised of molecules having a precisely definedstructure, it is more difficult to maintain uniform polymer properties,drug delivery properties, and therapeutic efficacy. As a result, it isrelatively difficult to obtain FDA approval of the linear polymer-drugcomposites. Another disadvantage with the use of linear polymers asdrug-carriers is that the location, and hence the availability, of thedrug is difficult to control. In particular, the drug can becomepermanently bound within the polymer, making the drug unavailable forits intended therapeutic purpose. The tendency of the drug to becomeburied in the linear polymer leads to greater unpredictability onaccount of the non-uniform or heterogenous properties of the linearpolymer molecules, and results in reduced drug efficiency because asignificant proportion of the drug molecules are not effectivelypresented to the cell being treated.

Accordingly, it would be highly desirable to provide a precisely defineddrug delivery system for platinum containing anti-tumor agents whichexhibits high drug efficiency, high drug carrying capacity, good watersolubility, good stability on storage, reduced toxicity, and improvedanti-tumor activity in vivo.

SUMMARY OF THE INVENTION

This invention pertains to dendritic polymer conjugates which are usefuldrug delivery systems for carrying platinum containing anti-tumor agentsto malignant tumors. The invention also pertains to methods of treatingmalignant tumors using the dendritic polymer conjugates, and to a methodof preparing a dendritic polymer platinate useful for carrying platinumcontaining anti-tumor agents to malignant tumors.

The dendritic polymer platinates of this invention comprise a dendriticpolymer conjugated to a platinum containing compound. The dendriticpolymer platinates are prepared by obtaining a dendritic polymer havingfunctional groups which are accessible to a platinum containing compoundcapable of interacting with the functional groups, contacting thedendritic polymer with the platinum containing compound, and reactingthe dendritic polymer with the platinum containing compound. Thedendritic polymer platinates are administered to an animal having amalignant tumor in an amount which is effective to inhibit growth of themalignant tumor, preferably intravenously, although other methods suchas oral, parental, subcutaneous or topical administration are alsoenvisioned.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of cationic dendrimers on hemolysisof rat erythrocytes;

FIG. 2 is a graph showing the effect of anionic dendrimers on hemolysisof rat erythrocytes;

FIG. 3 is a graph showing the effect of anionic dendrimers on B16F10cells;

FIG. 4 is a graph showing the effect of cationic dendrimers on B16F10cells;

FIG. 5 is a graph showing the effect of cationic dendrimers on CCRF-CEMcells;

FIG. 6 is a graph showing the effect of anionic dendrimers on CCRF-CEMcells;

FIG. 7 is a graph showing the effect of anionic dendrimers on HepG2cells;

FIG. 8 is a graph showing the effect of cationic dendrimers on HepG2cells;

FIG. 9 is a graph showing chloride release from cisplatin in water andduring reaction of cisplatin to a generation 3.5 polyamidoamine;

FIG. 10 is a structural representation of possible variations inplatinum binding to dendrimer;

FIG. 11 is a graph showing the release of cisplatin at two physiologicalpH conditions;

FIG. 12 is a graph showing the effect of cisplatin and dendrimerconjugate on Cor L23 cells in vitro;

FIG. 13 is a graph showing the effect of cisplatin and dendrimerconjugate on B16F10 cells in vitro;

FIG. 14 is a graph showing the effect of cisplatin and dendrimerconjugate on CCRF cells in vitro;

FIG. 15 is a bar graph showing the effect of intraperitoneal injectionof dendrimer-platinum conjugate treatment on intraperitoneally injectedtumors;

FIG. 16 is a bar graph showing the effect of dendrimer-platinumconjugate on established B16 melanoma;

FIG. 17 is a graph showing the accumulation of dendrimer-platinum andplatinum intravenously injected in C57 mice, bearing B16F10subcutaneously injected tumor;

FIG. 18 is a graph showing the effect of dendrimer on the body weight ofDBAZ mice bearing L1210 leukemia;

FIG. 19 is a bar graph showing the effect of dendrimer-platinumconjugates on established B16 melanoma; and

FIG. 20 is a series of plots which show the 48 hr. pharmacokinetics ofdendrimer-Pt and cisplatin in C57 mice bearing S.C. B16F10 tumor.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The dendritic polymers which may be used include generally any of theknown dendritic architectures including dendrimers, regular dendrons,controlled hyperbranched polymers, dendrigrafts, and randomhyperbranched polymers. Dendritic polymers are polymers with denselybranched structures having a large number of reactive groups. Adendritic polymer includes several layers or generations of repeatingunits which all contain one or more branch points. Dendritic polymers,including dendrimers and hyperbranched polymers, are prepared bycondensation reactions of monomeric units having at least two reactivegroups. The dendrimers which can be used include those comprised of aplurality of dendrons that emanate from a common core which can be asingle atom or a group of atoms. Each dendron generally consists ofterminal surface groups, interior branch junctures having branchingfunctionalities greater than or equal to two, and divalent connectorsthat covalently connect neighboring branching junctures.

Dendrons and dendrimers can be prepared by convergent or divergentsynthesis.

Divergent synthesis of dendrons and dendrimers involves a moleculargrowth process which occurs through a consecutive series ofgeometrically progressive step-wise additions of branches upon branchesin a radially outward molecular direction to produce an orderedarrangement of layered branched cells. Each dendritic macromoleculeincludes a core cell, one or more layers of internal cells, and an outerlayer of surface cells, wherein each of the cells includes a singlebranch juncture. The cells can be the same or different in chemicalstructure and branching functionality. The surface branch cells maycontain either chemically reactive or passive functional groups.Chemically reactive surface groups can be used for further extension ofdendritic growth or for modification of dendritic molecular surfaces.The chemically passive groups may be used to physically modifieddendritic surfaces, such as to adjust the ratio of hydrophobic tohydrophilic terminals, and/or to improve the solubility of the dendriticpolymer for a particular solvent.

Convergent synthesis of dendrimers and dendrons involves a growthprocess which begins from what will become the surface of the dendron ordendrimer and progresses radially in a molecular direction toward afocal point or core. The dendritic polymers may be ideal or non-ideal,i.e., imperfect or defective. Imperfections are normally a consequenceof either incomplete chemical reactions, or unavoidable competing sidereactions. In practice, real dendritic polymers are generally nonideal,i.e., contain certain amounts of structural imperfections.

The hyperbranched polymers which may be used represent a class ofdendritic polymers which contain high levels of nonideal irregularbranching as compared with the more nearly perfect regular structure ofdendrons and dendrimers. Specifically, hyperbranched polymers contain arelatively high number of irregular branching areas in which not everyrepeat unit contains a branch juncture. The preparation andcharacterization of dendrimers, dendrons, random hyperbranched polymers,controlled hyperbranched polymers, and dendrigrafts is well known.Examples of dendimers and dendrons, and methods of synthesizing the sameare set forth in U.S. Pat. Nos. 4,410,688, 4,507,466; 4,558,120;4,568,737; 4,587,329; 4,631,337; 4,694,064; 4,713,975; 4,737,550;4,871,779 and 4,857,599. Examples of hyperbranched polymers and methodsof preparing the same are set forth, for example in U.S. Pat. No.5,418,301.

The dendritic polymers or macromolecules useful in the practice of thisinvention are characterized by a relatively high degree of branching,which is defined as the number average fraction of branching groups permolecule, i.e., the ratio of terminal groups plus branch groups to thetotal number of terminal groups, branched groups and linear groups. Forideal dendrons and dendrimers, the degree of branching is 1. For linearpolymers, the degree of branching is 0. Hyperbranched polymers have adegree of branching which is intermediate that of linear polymers andideal dendrimers, a degree of branching of at least about 0.5 or higheris preferred. The degree of branching is expressed as follows:$f_{br} = \frac{N_{t} + N_{b}}{N_{t} + N_{b} + N_{1}}$

where N_(x) is the number of type x units in the structure. Bothterminal (type t) and branched (type b) units contribute to the fullybranched structure whilst linear (type 1) units reduce the branchingfactor; hence

 0≦f_(br)≦1

where f_(br)=0 represents the case of a linear polymer and f_(br)=1represents the case of a fully branched macromolecule.

Dendritic polymers suitable for use with the invention also includemacromolecules commonly referred to as cascade molecules, arborols,arborescent grafted molecules, and the like. Suitable dendritic polymersalso include bridged dendritic polymers, i.e., dendritic macromoleculeslinked together either through surface functional groups or through alinking molecule connecting surface functional groups together, anddendritic polymer aggregates held together by physical forces. Alsoincluded are spherical-shaped dendritic polymers and rod-shapeddendritic polymers grown from a polymeric core.

The dendritic polymers used in the practice of this invention can begenerationally monodisperse or generationally polydisperse. Dendriticpolymers in a monodisperse solution are substantially all of the samegeneration, and hence of uniform size and shape. The dendritic polymersin the polydisperse solution comprise a distribution of differentgeneration polymers. The dendritic polymer molecules which may be usedin the practice of this invention include mixtures of different interiorand exterior compositions or functionalities. Examples of suitabledendritic polymers include poly(ether) dendrons, dendrimers andhyperbranched polymers, poly(ester) dendrons, dendrimers andhyperbranched polymers, poly(thioether) dendrons, dendrimers andhyperbranched polymers, poly(amino acid) dendrons dendrimers andhyperbranched polymers, poly(arylalkylene ether) dendritic polymers andpolypropylamine dendrimers, dendrimers and hyperbranched polymers.Poly(amidoamine) (PAMAM) dendrimers have been found to be particularlyuseful for preparing the metal-containing complexes of this invention.

Dendritic polymers which are useful in the practice of this inventioninclude those that have symmetrical branch cells (arms of equal length,e.g., PAMAM dendrimers) and those having unsymmetrical branch cells(arms of unequal length, e.g. lysine-branched dendrimers) brancheddendrimers, cascade molecules, arborols, and the like.

The term “dendritic polymer” also includes so-called “hypercomb-branched” polymers. These comprise non-crosslinked poly-branchedpolymers prepared by (1) forming a first set of linear polymer branchesby initiating the polymerization of a first set of monomers which areeither protected against or non-reactive to branching and grafting,during polymerization, each of the branches having a reactive end unitupon completion of polymerization, the reactive end units beingincapable of reacting with each other; (2) grafting the branches to acore molecule or core polymer having a plurality of reactive sitescapable of reacting, with the reactive end groups on the branches; (3)either deprotecting or activating a plurality of monomeric units on eachof the branches to create reactive sites; (4) separately forming asecond set of linear polymer branches by repeating step (1) with asecond set of monomers; (5) attaching the second set of branches to thefirst set of branches by reacting the reactive end groups of the secondset of branches with the reactive sites on the first set of branches,and then repeating steps (3), (4) and (5) above to add one or moresubsequent sets of branches. Such hyper comb-branched polymers aredisclosed in European Patent Publication 0473088A2. A representativeformula for such hyper comb-branched polymer is:

wherein C is a core molecule; each R is the residual moiety of aninitiator selected from a group consisting of free radical initiators,cationic initiators, anionic initiators, coordination polymerizationinitiators and group transfer initiators; A and B are polymerizablemonomers or comonomers capable of withstanding the conditions requiredfor branching therefrom or grafting thereto, at least during thepolymerization of the {(A)—(B)} linear polymer chain and during itsgrafting to a prior {(A)—(B)} branch of the {(A)—(B)} core branch; eachG is a grafting component, and the designation

indicates that G can attach to either an (A) unit or a (B) unit; n isthe degree of polymerization of the indicated generation comb-branches;y is the fraction of B units in the indicated generation branch, and hasa value of 0.01 to 1; the superscripts 0, 1 and i designate thecomb-branch generation level, with i beginning at “2” and continuing forthe number of reiterative branch set generations in the polymer; and atleast n⁰ and n′ are ≧2.

For purposes of clarifying terminology, it should be noted that densestar dendrimers are built by reiterative terminal branching, while hypercomb-branched dendrimers are built by reiterative comb-branching. Indense star dendrimers, subsequent generation branches are attached tothe terminal moieties of a previous generation, thus limiting the degreeof branching to the functionality of the previous generation terminalmoiety, which would typically be two or three. In contrast, by branchingoligomers upon prior generation oligomer branches in accordance withhyper comb-branched dendrimer, one can dramatically increase the degreeof branching from generation to generation, and indeed can vary thedegree of branching from generation to generation.

The dendritic polymers which are believed to be most useful in thepractice of this invention are approximately monodispersed. That is,dendritic polymers in a monodispersed solution in which all of thedendritic polymer molecules are substantially of the same generation,and hence of uniform size and shape, are preferred. Monodispersedsolutions of dendrimers are particularly preferred.

The dendritic polymers used in the practice of this invention haveterminal functional groups which are accessible to a platinum containingcompound which is capable of interacting with the functional groups.Dendritic polymers having anionic functional groups are preferred.Examples of anionic function groups include sulfonates, sulfates andcarboxylate groups, with carboxylate groups being particularlypreferred.

Examples of suitable dendritic polymers which may be used in thepractice of this invention include polyamidoamine dendrimers, especiallycarboxylate terminated polyamidoamine dendrimers, and carboxylateterminated polypropylamine dendrimers.

The generation of the dendritic polymer, and hence the size of thedendritic polymer, which may be utilized in the practice of thisinvention may vary considerably. For example, generation 3.5polyamidoamine dendrimers are acceptable for use in the practice of thisinvention. However, higher and lower generations are also expected to beuseful.

The platinum containing compound can be generally any platinumcontaining compound which can be reversibly conjugated to the functionalgroups of the dendritic polymer and which exhibits anti-tumor activitywhen released from the dendritic polymer. The preferred platinumcontaining compound is cisplatin (cis-diamminedichloroplatinum). Othersuitable platinum containing compounds include those having atetravalent platinum atom bonded to the nitrogen of two amine ligands,which may be the same or different, the amine ligands being in cisconformation with respect to each other, at least one of the remainingligands is capable of interacting with or being displaced by afunctional group of the dendritic polymer. An example of such compoundis cis-diamminedichloroplatinum. A large number of different analoguesof cisplatin have been investigated (see for example; J. Respondek andJ. Engel, Drugs Of The Future 1996, 21(4), 391-408 and R. B. Weiss andM. C. Christian, Drugs 1993, 46, 360-377) and many of these differentplatinum-derivatives are likely to be useful in the present invention.

The dendritic polymer platinates may be prepared by dissolving thedendritic polymer in a suitable solvent, such as water, contacting thedissolved dendritic polymer with a dissolved platinum containingcompound under conditions sufficient to cause the platinum containingcompound to react with the dendritic polymer to form a dendriticpolymer-platinate. A cisplatin to dendrimer (Generation 3.5 PAMAMdendrimer) molar ratio of 35:1 was used in the experiments described inthe examples and these conditions resulted in a compound that was foundto be 44 nm in diameter by GPC. The ratio of cisplatin molecules todendritic polymer molecules can vary considerably. Dendritic polymerplatinates having a cisplatin to dendritic polymer molar ratio of fromabout 100:1 to about 1:1 have been evaluated and are expected to providepractical advantages. The large size of this compound in comparison tothe dendrimer itself (4 nm) is likely due to the formation ofintermolecular bonds between the dendrimers which are mediated bycisplatin. It is likely that by changing the ratio of cisplatin, orother platinum analogues, to dendrimer that it would be possible toproduce materials which have different average sizes and alsopotentially different biological properties.

The dendritic polymer platinate may be administered to animals,especially humans, in a therapeutically effective quantity to treat amalignant tumor in the animal. The dendritic polymer platinates may beadministered orally or topically, but are preferably administeredparentally, such as by subcutaneous injection, intraperitonealinjection, intravenous injection or intramuscular injection. Aneffective amount of a generation 3.5 polyamidoamine dendrimer-cisplatinconjugate in which the cisplatin loading is about 25% by weight (i.e.,25% by weight of the conjugate is cisplatin) has been found to be fromabout 1 milligram per kilogram of body weight to about 15 milligrams perkilogram of body weight for a mouse (KBA2 or C57) for an intraperitonealinjection. Suitable quantities of various dendritic polymer-platinateswhich are therapeutically effective in the treatment of variousmalignant tumors in other animals can be determined through routineexperimentation and testing.

It is anticipated that the dendritic polymer-platinate compounds will beeffective in the treatment of various malignancies in which cisplatinand other platinum containing anti-tumor agents have been found to betherapeutically affective, including ovarian cancer, lung cancer,testicular cancer, breast cancer, stomach cancer and lymphoma, also itis anticipated that the dendritic polymer-platinate compounds could beused in combination therapy with other anticancer agents. In vitrotesting and in vivo testing on mice suggest that the dendriticpolymer-platinate compounds is also therapeutically effective in thetreatment of melanoma and human lymphoblastic leukemia.

Experimental Methods

Synthesis and Characrerisation

Polyamidoamine dendrimers (PAMAM) (Sigma) were synthesised according tothe method of Tomalia et al., Polymer J., 17(1985) 117-132 (4).Dendrimers generation 3.5 (COONa) and 4 (NH₂) were reacted withcisplatin under stirring at room temperature for 4 h during which time

Dendrimer Mol. Wt. No. funct. Generation (Daltons) groups 4.0 14,215 64(NH₂) 3.5 12,419 64 (COONa) chloride ion release was followed using achloride electrode. The dendrimer-platinum (Pt) was characterized usingthe OPDA (colorimetric) assay and AAS (total Pt), GPC (Mw and free Pt),IR and NMR.

Pt Release

To study the rate of Pt release and also dendrimer biodegradation theconjugate was incubated in buffers at pH 7.4 and 5.5 and also in thepresence of serum and lysosomal enzymes.

Biological Evaluation

In vitro cytotoxicity was assessed against B16F10 melanoma, CCRF (humanlymphoblastic leukemia) and Cor-L23 (human lung) cells using the MTTassay. Dendrimer-Pt and free cisplatin were administered i.p. (days1,2,3 or day 1 only) to DBA2 or C57 mice bearing i.p. inoculated L1210or B16F10 tumors (respectively). Alternatively drug was administeredi.v. to mice bearing s.c. B16F10 when the tumor reached palpable size(50-100 sqmm). Animal weight, tumor size and animal survival weremonitored (UK guidelines for animal experiments involving neoplasia werefollowed.)

Materials

Polyamidoamine (PAMAM) Starburst® dendrimers were purchased from Aldrich(UK) Ltd.

EXAMPLE 1 Effect of PAMAM Dendrimer on the Stability of Rat ErythrocytesIncubated in vitro

Method

Polyamidoamine dendrimers (cationic and anionic) of increasinggenerations were incubated with rat erythrocytes obtained from an adultwistar rat. The interaction of the dendrimer with the erythrocyte wasassessed spectrophotometrically by the detection of released hemoglobin,induced by lysis, with a spectrophotometer at 550 nm. Variousconcentrations of dendrimer, controls (methanol (BDH)), poly-L-lysine(HBr salt—56.5 KD Mw (Sigma)), and dextran (74 KD Mw (Sigma)) (dissolvedin physiologically buffered saline) were incubated with the raterythrocytes (2% w/v solution) for 1 h at 37° C., and at 10 rpm (shakingwater bath). On completion, the erythrocytes were spun in a centrifugeat 1500×g for 10 minutes to pellet the cells and 100 μl of thesupernatant was removed and analyzed on the spectrophotometer afterblanking against PBS. The results are expressed in FIGS. 1 and 2 as apercentage of hemoglobin release compared to an intrinsic control(Triton×100 (1% v/v solution (Sigma)) which gave 100% lysis.

Result

Cationic dendrimers, except generation 1, were lytic, whereas solubleanionic dendrimers (including PAMAM gen. 3.5) were not lytic.

EXAMPLE 2 Cell Cytotoxicity of Unmodified Dendrimers Against B16F10Cells

Method

B16F10 cells are an adherent murine melanoma cell line. B16F10 cellswere seeded at a density of 1×10⁵ cells per ml (1×10⁴ cells per well) ina 96 well flat bottomed microtiter plate (costar) in RPMI 1640 tissueculture media (Gibco) supplemented with 10% fetal calf serum (FCS)(Gibco). All cellular growth and cytotoxic incubations were carried outin a cell incubator at 37° C. and 5% CO₂ atmosphere.

Cell density was assessed using an improved neurenbrow hemocytometer(Sigma).

The cells were washed with PBS twice and fresh RPMI media (supplementedwith FCS) was added, and the cells were then seeded in a microtiterplate. The cells were left for 24 h to recover and readhere.

All polymers and controls were dissolved in RPMI media (supplementedwith FCS) and then sterilized through a 0.2 μm sterile filter(acrodisk), the first few microliters of the solution being discarded inthe case of adherence of the polymer to the filter membrane. Polymer andcontrols were then added in increasing concentrations to the cells inthe microtiter plate. Some cells were left in media only to act ascellular controls. The methanol and poly-L-lysine were negative controlsand the dextran was a positive control. The cells were left in theincubator for 72h, and checked occasionally for yeast or bacterialcontamination. 5h prior to the incubation time end point, at 67h, 20 μltetrazolium (colorimetric) dye (MT) was added and the cells left for thefinal 5h. Then cellular media was removed, 100 μl of optical grade DMSO(Sigma) was added and the MTT crystals dissolved. The plates were readin a Titerteck plate reader and the results (OD) are expressed in FIGS.3 and 4 as a percentage of the OD seen in cell wells containing nopolymer or control.

Result

Cationic dendrimers were cytotoxic (similar to poly-L-lysine) towardsthe cell line, while anionic dendrimers (including PAMAM gen. 3.5) werenot cytotoxic (similar to dextran).

EXAMPLE 3 Cell Cytotoxicity of Unmodified Dendrimers Against CCRF-CEMCells

Method

CCRF-CEM cells are lymphoblastic leukemia and a suspension cell line,i.e. it grows in suspension. CCRF-CEM cells were seeded at a density of5×10⁴ cells per ml (5×10³ cells per well) in a 96 well V-shapemicrotiter plate (costar) in RPMI 1640 tissue culture media (Gibco)supplemented with 10% fetal calf serum (FCS) (Gibco). All cellulargrowth and cytotoxic incubations were carried out in a cell incubator at37° C. and 5% CO₂ atmosphere.

Cell density was assessed using an improved neurenbrow hemocytometer(Sigma). The cells were centrifuged at 1000×g and resuspended in freshmedia (supplemented with FCS) before the cell density was assessed. Thecells were then seeded in a microtiter plate. The cells were left for 24h to recover and readhere.

All polymers and controls were dissolved in RPMI media (supplementedwith FCS) and then sterilized through a 0.2 μm sterile filter(acrodisk), the first few microliters of the solution being discarded inthe case of adherence of the polymer to the filter membrane. Polymer andcontrols were then added in increasing concentrations to the cells inthe microtiter plate. Some cells were left in media only to act ascellular controls. The methanol and poly-L-lysine were negative controlsand the dextran was a positive control. The cells were left in theincubator for 72 h, and checked occasionally for yeast or bacterialcontamination. 5 h prior to the incubation time end point, at 67 h, 20μl tetrazolium (colorimetric) dye (MTT) was added, and the cells leftfor the final 5 h. Then cellular media was removed, 100 μl of opticalgrade DMSO (Sigma) was added and the MTT crystals dissolved. The plateswere read in a Titerteck plate reader and the results (OD) are expressedin FIGS. 5 and 6 as a percentage of the OD seen in cell wells containingno polymer or control.

Result

Cationic dendrimers were cytotoxic (similar to poly-L-lysine) towardsthe cell line, while anionic dendrimers (including PAMAM gen. 3.5) werenot cytotoxic (similar to dextran).

EXAMPLE 4 Cell Cytotoxicity of Unmodified Dendrimers Against HepG2 Cells

Method

HepG2 is a hepatocellular carcinoma and is an adherent cell line, i.e.it grows in a monolayer. HepG2 cells were seeded at a density of 1×10⁵cells per ml (1×10⁴ cells per well) in a 96 well flat bottomedmicrotiter plate (costar) in Minimal Essential Medial (MEM) tissueculture media (Gibco) supplemented with 10% fetal calf serum (FCS)(Gibco). All cellular growth and cytotoxic incubations were carried outin a cell incubator at 37° C. and 5% CO₂ atmosphere.

Cell density was assessed using an improved neurenbrow hemocytometer(Sigma). The cells were washed with PBS twice and fresh RPMI media(supplemented with FCS) added, the cells were then seeded in amicrotiter plate. The cells were left for 24 h to recover and readhere.

All polymers and controls were dissolved in RPMI media (supplementedwith FCS) and then sterilized through a 0.2 μm sterile filter(acrodisk), the first few microliters of the solution being discarded inthe case of adherence of the polymer to the filter membrane. Polymer andcontrols were then added in increasing concentrations to the cells inthe microtiter plate. Some cells were left in media only to act ascellular controls. The methanol and poly-L-lysine were negative controlsand the dextran was a positive control. The cells were left in theincubator for 72 h, and checked occasionally for yeast or bacterialcontamination. 5 h prior to the incubation time end point, at 67 h, 20μl tetrazolium (colorimetric) dye (MTT) was added and the cells left forthe final 5 h. The cellular media was removed and 100 μl of opticalgrade DMSO (Sigma) was added and the MTT crystals dissolved. The plateswere read in a Titerteck plate reader and the results (OD) are expressedin FIGS. 7 and 8 as a percentage of the OD seen in cell wells containingno polymer or control.

Result

Cationic dendrimers were cytotoxic (similar to poly-L-lysine) towardsthe cell line, while anionic dendrimers (including PAMAM gen. 3.5) werenot cytotoxic (similar to dextran).

EXAMPLE 5 Synthesis of the Dendrimer-Platinate

Method

1 g of polyamidoamine Starburst® dendrimer generation 3.5 was dissolvedin double deionized water (DDW-10 ml). 0.8 g of cisplatin(cis-diamminedichloro platinate (II)) was dissolved in 400 ml of water(cisplatin maximum solubility is 2 mg/ml) (a molar ratio of cisplatin todendrimer of 35:1). Once the cisplatin was fully dissolved in the water,the dendrimer was added dropwise under stirring to the cisplatin. Thesolution was left to react for at least 4 h. Then the solution wastransferred to a dialysis bag (10 KD MW cut off) and dialyzed againstDDW for 2-3 days. The water was changed every few hours. Thedendrimer-platinate was transferred to a glass container and freezedquickly using liquid nitrogen before being lyophilized (VA Howe).

The above procedure was repeated but with varying molar ratios from1-100 in steps of 10. And the optimal ratio determined for the reaction.

Results

The weight percent was reproducibly determined at 25 wt %, while themaximum wt % achievable was approximately 40 wt %. The ratio experimentallowed speculation on the type of cisplatin binding.

EXAMPLE 6 Chloride Ion Release

Method

A chloride ion release meter (Jenway) was used to determine the reactionkinetics of the cisplatin and dendrimer reaction. A known amount ofcisplatin was reacted with a known amount of dendrimer and at specifictime intervals, 20 μl of the reaction mixture was removed and added tothe chloride meter and the chloride content determined. This isindicative of chloride ions leaving the cisplatin on reaction with thedendrimer or hydrolysis in water. A water control was also completed.The results are shown in FIG. 9

Result

The reaction time was found to be 4 h. The reaction kinetics of thecisplatin and dendrimer were much faster than hydrolysis alone.

EXAMPLE 7 Atomic Absorption Spectroscopy

Method

A known amount of dendrimer-platinate (typically 10 mg) was dissolved inDDW (250 ml), and standards of cisplatin or potassiumtetrachloroplatinate (II) were made up in the ppm range 1-100. A fewdrops of concentrated nitric acid (10 M (BDH)) were added to preventinterference. A Perkin-Elmer atomic absorption spectrophotometer wasused. The machine was set at the maximum PPM and calibrated on the PPMrange. The dendrimer-platinate (unknown) was then analyzed and acalibration curve constructed. The content of platinum was assessed andexpressed as a weight percentage.

Result

Typically the dendrimer platinate contained 25 wt % platinum.

EXAMPLE 8 Nuclear Magnetic Resonance Spectroscopy (NMR), Gel PermeationChromatography (GPC) and Particle Sizing by Photon CorrelationSpectroscopy (PCS)

Method

Samples of dendrimer-platinate were analyzed using NMR (Bruker 400 MHz)using 1H, 13C, HCOSY, HCCOSY. Gel permeation chromatography was used toanalyze the sample on G2000 and G400PW columns (Supelco) linked inseries with refractometer (RI (Gilson)) and UV-Vis spectrophotometerdetection (Severn). The pump flow rate was set to 1 ml/min. The RI rangewas typically set to 2-4 AU, and the UV-Vis detection wavelength was setto the UV absorbance of dendrimer-platinate solution (279 nm). Themobile phase used were water, PBS and high salt (0.25 NaCl). The columnswere calibrated using pullulan and protein standards. Dendrimergeneration 3.5 and dendrimer-platinate were analyzed by PCS in DDW.

Result

The NMR confirmed surface conjugation of the platinate on the dendrimer,through chemical shift enhances in key resonances relating to carboxygroups. The GPC showed the presence of a number of species whichappeared to be dendrimer crosslinked by the platinum, with potentiallythe presence of mono and dimeric dendrimers as well. The particle sizefor the dendrimer was approximately 4 nm and the dendrimer-platinate 44nm. Several possible modes of platinum binding to dendrimer are shown inFIG. 10.

EXAMPLE 9A In Vitro Release of Platinum from the Dendrimer-Platinate inBiological Fluids

Method

Known amounts of cisplatin and dendrimer were placed in two bufferedsolutions (PBS at pH 7.4 and Citrate-Phosphate at pH 5.5) to simulatedifferent biological compartments (the plasma/extracellular and thelysosomal compartments, respectively). The solution was sealed in adialysis bag with a molecular cut off of 10 KD. Then the bag was placedin a container filled with the respective buffered solution. Thesolutions were then placed in heated water bath at 37° C. At regularintervals, samples from the buffer solutions were removed and analyzedin triplicate (over a period of 74 h). At the end of the experiment, asample was taken from within the bag. All the samples were analyzedusing atomic absorption spectroscopy as described previously.

Result

The amount of platinum released at pH 5.5 was slightly greater than thatreleased at pH 7.4. However, as shown in FIG. 11, the total amountreleased over time remained less than 1% of the total.

EXAMPLE 9B Cell Cytotoxicity of Dendrimer-Platinate (B16F10, L1210,CorL23)

Method

Cells were seeded at a density of 1×10⁵ cells per ml (1×10⁴ cells perwell) in a 96 well flat bottomed microtiter plate (costar) in RPMI 1640tissue culture media (Gibco) supplemented with 10% fetal calf serum(FCS) (Gibco). All cellular growth and cytotoxic incubations werecarried out in a cell incubator at 37° C. and 5% CO₂ atmosphere.

Cell density was assessed using an improved neurenbrow hemocytometer(Sigma). The cells were washed with PBS twice and fresh RPMI media(supplemented with FCS) added, the cells were then seeded in amicrotiter plate. The cells were left for 24 h to recover and readhere.If cells were in a suspension they were spun at 1000×g and resuspendedin fresh media.

The dendrimer-platinate and cisplatin were dissolved in RPMI media(supplemented with FCS) and then sterilized through a 0.2 μm sterilefilter (acrodisk), the first few microliters of the solution beingdisguarded in the case of adherence of the polymer to the filtermembrane. Then dendrimer and cisplatin were added in increasingconcentrations to the cells in the microtiter plate. Some cells wereleft in media only to act as cellular controls. The cells were left inthe incubator for 72 h, and checked occasionally for yeast or bacterialcontamination. 5 h prior to the incubation time end point, at 67 h, 20μl tetrazolium (colorimetric) dye (MTT) was added and the cells left forthe final 5 h. Then cellular media was removed and 100 μl of opticalgrade DMSO (Sigma) was added and the MTT crystals dissolved. The plateswere read in a Titerteck plate reader and the results (OD) are expressedin FIGS. 12, 13 and 14 as a percentage of the OD seen in cell wellscontaining no dendrimer-platinate or cisplatin.

Result

The dendrimer-platinate was less cytotoxic than the cisplatin alone byseveral orders of magnitude.

EXAMPLE 10 Pharmacology (i.p. Tumor Verses i.p. Injection)

Method

L1210 or B16F10 cells were injected at a cell density into a mouse (DBA2or C57 respectively, 25g) at a cell density of 1×10⁵ (0.9% salinesolution) into the intraperitoneal (i.p. 100 μl) cavity. 24 h later, thedendrimer-platinate and cisplatin (on one day or on three consecutivedays) were injected at a concentration according to the weight of themouse (e.g. 1 mg/kg-15 mg/kg). The mouse body weight and generaltoxicity was also monitored according to UK guidelines in the use ofanimals used in neoplasia studies. At the end point the gross morphologyof the organs was noted.

Result

This pharmacology demonstrated the maximum tolerated does of thedendrimer-platinate (25-50 mg/kg). As shown in FIG. 15, I.P. delivery ofdendrimer platinate showed anti-tumor activity but not substantiallybetter than cisplatin alone.

EXAMPLE 11 Pharmacology (s.c. Tumor Verses i.v. Injection)

Method

B16F10 cells were injected at a cell density of 1×10⁵ (0.9% salinesolution) into the left or right flank of the C57 mouse subcutaneously(s.c.). The mouse was then left until the tumor was visible at apalpable size of between 50-100 sqmm. Then the dendrimer-platinate andcisplatin were injected intravenously (i.v.) into the tail vein at therespective doses. The animal was monitored and the tumor size measuredusing calipers and recorded on a daily basis. When the animal tumor sizewas between 300-400 sqmm, the animal was culled. The tumor excised andweighed and gross morphology of the organs noted.

Result

The dendrimer-platinate was active against the s.c. tumor anddemonstrated a significant difference in the final tumor weight andsurvival time, as shown in FIG. 16.

EXAMPLE 12 Biodistribution of Dendrimer-Platinate in Vivo

Method

C57 mice were injected subcutaneously with B16F10 cells at a celldensity of 1×10⁵ cells per mouse. The tumor was allowed to reach apalpable size before injecting the dendrimer-platinate or cisplatin i.v.At specific time points (0-24 h) the animal was culled and key organs(liver, kidney, and blood) including the tumor were isolated andweighed. The organs were solubilized in concentrated nitric acid (10)and hydrogen peroxide added to decolorize the solution during boiling.The solutions were made up to a fixed volume (25 ml) and then analyzedusing atomic absorption spectroscopy after addition of lanthanum(excess) to free up bound platinum.

Result

Compared to cisplatin alone, the dendrimer-cisplatin was found toaccumulate preferentially in the tumor by at least 3×, relativelyquickly after the injection. The results are shown in FIG. 17.

EXAMPLE 13 Measurement of the Pharmacokinetics of Cisplatin andDendrimer-Platinate In Vivo

Method

B16F10 cells (10⁵ cells) were injected into C57 mice s.c. to provide asolid tumor model. When the tumor developed to a mean area of 50-100 mm²(after approximately 12 days) animals were injected i.v. with a singledose of cisplatin (1.0 mg/kg, at is maximum tolerated dose) ordendrimer-Pt (1 or 15 mg/kg). In both cases animals were monitored forgeneral health and weight loss. At time points 0, 1, 5, 12, 24 and 48 hmice (5 per group) were culled. Blood and tissue samples were taken. Theorgans were digested in nitric acid (10 ml, 10 M) under heating (boilingfor 2 days). Hydrogen peroxide was added to a known volume to oxidisethe solution and the Pt concentration determined by graphite AAS.

Result

The tumor area under the curve (AUC) for accumulation of dendrimerplatinate was 5 fold (dendrimer-Pt 1 mg/kg) and 50 fold (dendrimer-Pt 15mg/kg) higher than seen for cisplatin (1 m/kg). Accumulation at sites oftoxicity (kidney) were reduced.

Summary Of Body Distribution Data AUC value (μg Pt/mL blood or μgPt/organ) over 48 h Cisplatin Dendrimer-Pt Dendrimer-Pt Organ 1 mg/kg 1mg/kg 15 mg/kg Tumor 5.3 25.4 264.9 Blood 9.4 10.7 502.0 Liver 51.6 17.0193.2 Kidney 57.6 138.1 244.2 Ratio of AUC Values Ratio Ratio AUCDendrimer-Pt AUC Dendrimer-Pt (1 mg/kg)/ (15 mg/kg)/ Organ AUC Cisplatin(1 mg/kg) AUC Cisplatin (1 mg/kg) Tumor 4.8 50.0 Blood 1.1 53.4 Kidney2.4 4.2 Liver 0.3 3.7 Ratio of AUC values obtained in terms ofTumor/Blood, Tumor/Liver or Tumor/Kidney Cisplatin Dendrimer-PtDendrimer-Pt Ratio 1 mg/kg 1 mg/kg 15 mg/kg Tumor/ 0.56 2.37 (4x) 0.53(same) Blood Tumor/ 0.09 0.18 (2x) 1.08 (12x) Kidney Tumor/ 0.10  1.49(15x) 1.37 (14x) Liver

Results and Discussion

Dendrimer-Pt Cisplatin interacted with the carboxy surface of gen. 3.5giving a conjugate with 25 wt % Pt loading which was highly watersoluble and stable on storage. Lack of interaction of Pt with dendrimergen. 4 indicates that Pt does not simply become entrapped in thedendrimer core or react with terminal primary amino groups.Stoichiometry indicates interaction is generally bifunctional, but GPCshowed some evidence for intramolecular crosslinking. IR and NMRconfirmed Pt interaction at the dendrimer surface. Pt was found to betightly bound with very little Pt release over 72 h in vitro.

In vitro Evaluation Dendrimer gen. 3.5 undergoes slight degradation inphysiological buffers over 24 h, increasing with pH (5.5-7.4), and isalso degraded in strong acid.

The dendrimer-Pt displayed anti-tumor activity against CCRF and Cor L23but in both cases was less active than cisplatin; this was expected dueto the different mechanism of cellular uptake. Up to concentrations of 2mg/ml (Pt-equiv.) the dendrimer-Pt was inactive against B16F10 melanomain vitro (see FIG. 19).

IC₅₀ Values (μ/ml, Pt-equiv.)) Cell line Cisplatin Dendrimer-Pt B16F109 >2000 CCRF-CEM 5    520 Cor L23 1    380

In vivo Evaluation The dendrimer-Pt showed anti-tumor activity in allthe tumor models tested, including the platinum resistant B16F10 model.It was confirmed that the generation 3.5 dendrimer displayed neitherinherent anti-tumor activity or general toxicity (see FIG. 18).

Activity against L1210 ip (doses ip days 1, 2, 3) Dose (Pt- ToxicTreatment equiv mg/Kg) T/C deaths cisplatin  2   171 0/10 cisplatin  3   64 9/10 dend-Pt  2 >123 0/5 dend-Pt 10 >132 0/5 dend-Pt 15 >132 0/5

Activity against BL6F10 ip (dose day 1) Dose (Pt- Toxic Treatment equivmg/kg) T/C deaths cisplatin  5  89 2/5 dend-Pt  5 105 0/5 dend-Pt 10 1080/5 dend-Pt 15 129 5/5^(T) ^(T)chronic toxicity

Anti-tumor activity was more pronounced against sensitive tumors e.g.L1210 and in the case of s.c. tumors with i.v. administration of drug.

Conclusion

Dendrimer-Pt has greater water solubility than cisplatin, was 3-5 timesless toxic, anti-tumor activity in vivo and was found to preferentiallyaccumulate in tumor tissue.

What is claimed is:
 1. A method for treatment of cancerous tumors inmammals, comprising administering a therapeutically effective quantityof a dendritic polymer-platinate compound to a mammal having a canceroustumor.
 2. The method of claim 1, wherein the dendritic polymer is adendrimer.
 3. The method of claim 1, wherein the dendritic polymer hasanionic functional groups.
 4. The method of claim 3, wherein the anionicgroups are carboxylate groups.
 5. The method of claim 1, wherein thedendritic polymer is a polyamidoamine dendrimer.
 6. The method of claim1, wherein the dendritic polymer is polypropylamine.
 7. The method ofclaim 1, wherein the platinum containing compound is a compoundcomprising a central tetravalent platinum atom bonded to the nitrogenatom of two amine ligands, which may b the same or different, the amineligands being in cis confirmation with respect to each other and atleast one of the remaining ligand sites is coupled to the dendriticpolymer.
 8. The method of claim 1, wherein the platinum containingcompound is cisplatin.
 9. The method claim 1, wherein the dendriticpolymer-platinate compound is administered parentally.
 10. The method ofclaim 1, wherein the dendritic polymer-platinate compound isadministered intravenously or intraperitoneally.
 11. The method of claim1, wherein the dendritic polymer-platinate compound is administeredorally.
 12. The method of claim 1, wherein the dendriticpolymer-platinate compound is administered topically.
 13. The method ofclaim 1, wherein the dendritic polymer-platinate compound isadministered intraperitoneally.
 14. The method of claim 1, wherein themolar ratio of cisplatin to dendritic polymer in the conjugate is fromabout 100:1 to about 1:1.
 15. The method of claim 1, wherein the molarratio of cisplatin to dendritic polymer in the conjugate is about 35:1.